Roca‑Pinilla et al.

Microbial Cell Factories

https://doi.org/10.1186/s12934-022-01991-2
(2022) 21:267
Microbial Cell Factories

REVIEW Open Access

The future of recombinant host defense

peptides
Ramon Roca‑Pinilla1, Leszek Lisowski1,2, Anna Arís3* and Elena Garcia‑Fruitós3*

Abstract
The antimicrobial resistance crisis calls for the discovery and production of new antimicrobials. Host defense peptides
(HDPs) are small proteins with potent antibacterial and immunomodulatory activities that are attractive for transla‑
tional applications, with several already under clinical trials. Traditionally, antimicrobial peptides have been produced
by chemical synthesis, which is expensive and requires the use of toxic reagents, hindering the large-scale develop‑
ment of HDPs. Alternatively, HDPs can be produced recombinantly to overcome these limitations. Their antimicrobial
nature, however, can make them toxic to the hosts of recombinant production. In this review we explore the different
strategies that are used to fine-tune their activities, bioengineer them, and optimize the recombinant production of
HDPs in various cell factories.
Keywords: Host defense peptides, Antimicrobial proteins, Antimicrobial resistance, Inclusion bodies, Recombinant
production

Background resist bacterial infections thanks to the action of cecropin

In 2019 alone, 1.27 million people died globally due to [5]. Another example is the potent antimicrobial activ-
antimicrobial-resistant bacteria (ARB) [1]. At the current ity of rabbit neutrophils due to defensins [6] and the skin
rate of resistance development, 10 million people will die wound-healing abilities of an African clawed frog, thanks
by 2050 due to our inability to treat infections [1]. There to secreted magainins [7]. Since then, the field of HDPs
is, therefore, a severe need to find suitable alternatives as exploded and today there are more than 3000 known
effective as conventional antibiotics or that can be used peptide sequences that come from all domains of life,
in a combinatorial treatment [2]. including HDPs [8].
One potential alternative is the use of host defense The characteristics that usually define HDPs are their
peptides (HDPs), which are a diverse and well-studied short amino acidic sequences (between 12 and 50 amino
class of bioactive peptides (AMPs) that all multicellu- acids) [4], a net positive charge [5], a certain degree of
lar organisms produce as a defense mechanism against hydrophobicity [9] and a wide range of broad-spectrum
pathogenic microbes [3, 4]. HDPs were discovered in the biological activities [10]. Among these activities, HDPs
1980s thanks to the keen eye of researchers that could have microbicidal (effective against bacteria, virus, and
not explain what they observed with their current under- fungi) [11–13], antibiofilm [14, 15], and immunomodula-
standing of immunity. For instance, Cecropia moth pupa tory activities [16–18]. Interestingly, it might be difficult
that lacked antibodies or lymphocytes were still able to for microorganisms to develop resistance against HDPs
because of their multiple modes of action, which may
ultimately lead to microbial death [3].
*Correspondence: anna.aris@irta.cat; elena.garcia@irta.cat Their well-known characteristics make them ame-
3
Department of Ruminant Production, Institut de Recerca i Tecnologia nable to engineering [19–21] (Fig. 1A, B), peptide
Agroalimentàries IRTA​, 08140 Caldes de Montbui, Spain repurposing, such as engineered venoms that can be
Full list of author information is available at the end of the article modified to become non-toxic HDPs [22] (Fig. 1A),

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Roca‑Pinilla et al. Microbial Cell Factories (2022) 21:267 Page 2 of 15

Fig. 1 Schematic figure showing several strategies that can be used to engineer antimicrobial peptides. A Rational modification of peptides to
improve some of their features; B multidomain proteins based on the combination of different HDPs; C design new peptides based on their known
properties; or D find encrypted peptides “buried” in known protein sequences

development of multidomain proteins based on the including potential toxicity, low stability, half-life, and a
combination of different HDPs (Fig. 1B), de novo high production cost.
designs [9, 23] (Fig. 1C), or aid in the discovery of Together with the need to be safe and effective, one of
hidden peptides within larger protein structures [24] the big questions that remains is how to produce them in
(Fig. 1D). All these new technologies yield an almost large quantities in a sustainable way and with a competi-
unlimited potential to modify known sequences or dis- tive price. Besides, some strategies including chemical
cover new peptides and modes of action. modifications and delivery vehicles are being studied to
There are multiple ways to classify HDPs, considering improve the properties of these peptides [28].
their secondary structure or common ancestry, for exam- The first HDPs were isolated and purified from their
ple. In vertebrates, there are two major families of HDPs: natural sources, but this process is difficult and time-
cathelicidins and defensins [10]. The latter have a com- consuming, and results in low yields, making it diffi-
mon β-sheet core stabilized by three disulphide bridges. cult to scale up (Fig. 2). Most of the studies to date use
Depending on how the cysteine residues link together, chemically synthesized HDPs [29], and, by automated
defensins are classified into α-, β-, and θ-defensins [10]. solid-phase peptide synthesis (SPPS) [29]. This strategy
Instead, over one third of the cathelicidins are α-helical. involves the repetitive binding of different amino acids to
Cathelicidins are produced as prepropeptides that need obtain the desired peptide sequence, which is bound to a
to be secreted and then cleaved by serine proteases [25]. resin support. Once the sequence is obtained, the peptide
However, there are other families, such as histatins, can be retrieved from the solid support with high purity
which are histidine rich HDPs from mammals’ saliva via a cleavable linker.
(Fig. 1D) [10, 24, 26]. Yet, in addition to the high production costs, one of
Although some peptide-based antimicrobials are in the main problems of chemical synthesis is its environ-
advanced clinical trials, none of them has been granted mental impact, due to the excessive use of organic sol-
regulatory approval [27]. There are many reasons, vents during the process [30]. Thus, it does not suit the
beyond the scope of this review, as to why that is the case, needs of large-scale production [31]. Besides, chemical

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Roca‑Pinilla et al. Microbial Cell Factories (2022) 21:267 Page 3 of 15

Fig. 2 Advantages and disadvantages of different recombinant expressions systems to produce HDPs [52, 108–110]. Only the most utilized systems
are shown, as there are very few instances where mammalian cells have been used for recombinant HDP production, as it is probably too expensive
and unnecessary in many cases. The bulk of the work with plant systems is based on transgenic plants to enhance their properties with genetic
modifications, but not necessarily recombinant HDP production per se

synthesis can be tricky for longer peptide sequences Cell factories for recombinant HDP production
of more than 35 amino acid residues [32]. In this con- As an alternative to chemical synthesis, technologies
text, our goal in this review is to discuss the feasibil- based on recombinant DNA have been explored for the
ity of recombinant HDP production as an alternative to biosynthesis of HDPs. The recombinant production of
chemical synthesis, considering several of the microbial these peptides offers a more flexible, sustainable, scal-
cell factories that have been used so far, as well as vari- able, and cost-effective production [31]. Many organisms
ous protein forms and strategies that lead to success in can be used as hosts for recombinant HDP production,
recombinant HDP production endeavors. including plants, insect cells, mammalian cells, yeast,

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Roca‑Pinilla et al. Microbial Cell Factories (2022) 21:267 Page 4 of 15

and bacterial cells [33]. However, choosing the optimal Exploiting all of these, a group from MIT [43] expressed
expression organism is critical to ensure proper protein apidaecin, an insect HDP, fused to human serum albumin
yields, biological function, and final cost (Fig. 2). (HSA) in the yeast Pichia pastoris. They obtained yields
of more than 700 mg/L with no cell lysis and no debris
Recombinant production in bacteria removal steps required. However, because it is a fusion
The most extensively used host for recombinant expres- construct, downstream cleaving of the fusion tag was
sion of proteins and peptides are bacteria, as they are necessary. Fish [44], human [45] and fungal [46] peptides
easy to manipulate, grow fast and use inexpensive media. have also successfully been produced recombinantly in P.
Nevertheless, bacteria, have a limited ability to make pastoris (Table 1), showing evidence that it can be a use-
disulphide-bonds, glycosylation, and other post transla- ful alternative to the most used expression system for
tional modifications (PTMS) [34–36]. These limitations, recombinant production (E. coli). Saccharmoyces cerevi-
however, might not be critical for HDP recombinant pro- siae is another yeast-based system that has been used for
duction [37]. In the absence of a strict need for PTMS, the expression of HDPs [47, 48], albeit less than P. pasto-
the heterologous expression in bacteria is a reasonable ris. The yields (mg/L) for HDPs expressed in yeast found
approach for their production with sufficient conforma- in the relevant literature are highly variable [39], between
tional and functional quality. less than 0.1 to up to 831 mg/L, suggesting the need to
As it occurs for many other recombinant proteins, the fine tune the expression system for each of the peptides.
most utilized bacterium for HDP production is E. coli
(Table 1), since it has been widely studied as a recombi- Recombinant production in fungi
nant host, with an extensive knowledge of its genetics, The use of filamentous fungi for HDP production is still
biochemistry, and physiology [38]. There are well-estab- in its infancy. However, interest in their use is growing
lished protocols and a large catalogue of expression vec- due to their successful application in the recombinant
tors, and besides, it grows fast. In addition, some HDPs production of non-antimicrobial proteins, their abil-
have been successfully expressed in Lactococcus lactis ity to perform PTMS, scale-up ability, and inexpensive-
(Table 1) and Bacillus subtilis. Although there are only ness of culture. The fungal defensin plectasin, developed
few examples of HDPs produced in these LPS-free bac- for the treatment of Gram-positive bacterial infections
teria, they are appealing candidates considering their is produced recombinantly using Aspergillus oryzae as a
status as generally regarded as safe (GRAS). Most HDPs high efficiency recombinant protein expression system,
expressed in bacteria are produced with inducible expres- produced as a secreted product (Table 1) [49]. Another
sion systems yielding quantities that range between 2 and example is the recombinant production of a hybrid HDP
600 mg/L [39]. of magainin II-cecropin B, successfully expressed in
Cordyceps militaris with a yield of 3.86 mg/g of mycelium
Recombinant production in yeasts (Table 1) [50].
Sometimes, the production of cysteine rich cationic
HDPs fails in bacterial systems such as E. coli, due to an Recombinant production in insect cells
inefficient formation of disulphide bridges that leads to The first isolated HDP was cecropin (1980), an insect
improper folding and lack of bioactivity [40]. Another HDP [5]. Insect cell-based systems prove to be very valu-
concern when expressing HDPs in bacteria is their natu- able for the recombinant expression of HDPs. In gen-
ral lethality towards the host [41]. Therefore, switching to eral, the most prevalent systems are based on Drosophila
eukaryotic cells can be a suitable alternative to overcome melanogaster cell lines, although there is some work in
this challenge, allowing for the heterologous expres- mosquito and moth cells [51]. Two main approaches are
sion of HDPs. Yeasts are one of the simplest eukaryotic used to express HDPs in insect systems, either by using
organisms [42] and offer a good compromise between a Baculovirus gene expression system [35], or by stably
the higher complexities of eukaryotic cells and the rela- expressing a HDP by means of genome integration of the
tively simple and inexpensive recombinant production HDP-coding gene.
of prokaryotic systems (Fig. 2). Besides, they grow faster Even though they are more expensive than yeast and
than typical mammalian recombinant hosts such as prokaryotic-based systems, they offer PTMS such as gly-
Chinese Hamster Ovary (CHO) or Human Embryonic cosylation that might aid the proper structure and bioac-
Kidney (HEK293) cells. And yeasts are free from endo- tivity of HDPs [52], although it is not clear if PTMS are
toxins and harmful human viruses and can secrete large necessary in all instances, or at all, suggesting that their
amounts of heterologous recombinant proteins with lit- need (or their lack of ) might require to be studied on a
tle host cell protein secretion, which can simplify down- case-by-case basis. However, when the Baculovirus sys-
stream purification (Fig. 2). tem is used, the lytic cycle of the virus can cause large

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Roca‑Pinilla et al. Microbial Cell Factories (2022) 21:267 Page 5 of 15

Table 1 Recombinant expression of HDPs and the different cell hosts used to express them
HDP Type Family Host Protein ­forma Fusion tag References

Apidaecin Insect AMP L. lactis subsp. cremoris Secreted – [111]

NZ9000
Apidaecin Insect AMP Pichia pastoris Secreted Human serum albumin [43]
(HSA)
Big defensins Molusc big defensin E. coli BL21(DE3) Solubilized – [112]
Bovine lingual antimicrobial Bovine defensin E. coli BL21(DE3) Soluble eGFP [37]
peptide (LAP) E. coli Origami B (DE3) Solubilized
Buforin II Histone H2A-derived AMP E. coli BL21 (DE3) Solubilized Cystein-rich acidic peptide [92]
(CAP)
Cathelicidin-BF (CBF) Cathelicidin B. subtilis WB800 N Secreted Intein [95, 157]
SUMO
CcDef2gene Insect defensin E. coli BL21(DE3) Solubilized 3xCcDef2 Casette [113]
Cecropin Cathelicidin P. pastoris Soluble Oleosin [114]
ChMAP-28, mini- Goat cathelicidins E. coli BL21(DE3) Solubilized Trx [115]
ChBac7.5Nα, and mini-
ChBac7.5Nα
CRAMP Cathelicidin E. coli BL21(DE3) Soluble SUMO [41]
Cryptidin-2 Defensin E. coli Rosetta-gami B (DE3) Soluble Trx [64]
E5 and E6 Bovine bactenecin deriva‑ E. coli BL21(DE3) Soluble SUMO [41]
tive
Egyptian maize defensin Plant defensin E. coli BL21(DE3) Soluble GST [116]
(MzDef )
FaAMP Fungal defensin E. coli BL21 (DE3) Soluble eGFP [117]
fBD Flounder defensin E. coli BL21(DE3) Soluble – [118]
Fowlicidin-1 Chicken cathelicidin E. coli BL21 (DE3) Soluble Calmodulin [119]
Fungal defensin-like pep‑ Fungal defensin P. pastoris Secreted – [120]
tide (DLP)
GL13K Encrypted ­peptide a E. coli BLR Soluble Elastin-like recombinamers [121]
Gloverin Insect antibacterial protein Drosophila melanogaster S2 Secreted – [52]
Human neutrophil peptide Human defensin P. pastoris Secreted Polyhedrin-eGFP [45]
1 (HNP1)
Human neutrophil pep‑ Human defensin E. coli strain Soluble – [122]
tide-1 (HNP-1) XPX-1
Human α-defensin 5 (HD5) Human defensin E. coli BL21(DE3) Solubilized eGFP [20, 21, 37]
E. coli Origami B (DE3) Trx
Human α-defensin 5 (HD5) Human defensin P. pastoris Secreted Alpha-factor [123]
Human β-defensin 1 (HBD1) Human defensin E. coli AD202 Solubilized C-terminal fragment of light [124, 125]
Soluble meromyosin (LMM)
Trx
Human β-defensin 1 (HBD1) Human defensin S. cerevisiae AH22 Secreted – [126]
Human β-defensin 118 Human defensin E, coli Rosetta (DE3) Soluble – [127]
Human β-defensin 2 (HBD2) Human defensin E. coli BL21(DE3) Soluble Trx [125, 128–131]
Solubilized Keto-steroid isomerase (KSI)
Glutathione-S-transferase
(GST)
Human β-defensin 3 (HBD3) Human defensin E. coli BL21(DE3) Soluble Trx [119, 132]
Calmodulin
Human β-defensin 4 (HBD4) Human defensin E. coli BL21(DE3) Soluble Trx [133]
Human β-defensin 6 (HD6) Human defensin E. coli Origami (DE3) pLys Soluble Trx [134, 135]
Human β-defensin DEFB136 Human defensin E. coli BL21(DE3) Soluble Intein-chitin binding [136]
domain (CBD)
Hybrid peptide Cecropin Cecropin B. subtilis WB800N Secreted Small ubiquitin modifier [137]
AD (SUMO)
IDR-1 Innate defense regulator E. coli BL21(DE3) Soluble SUMO [41]
(IDR)

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Roca‑Pinilla et al. Microbial Cell Factories (2022) 21:267 Page 6 of 15

Table 1 (continued)
HDP Type Family Host Protein ­forma Fusion tag References

Indolicidin Cathelicidin E. coli BL21 (DE3) Soluble Calmodulin [119]

Insect defensin A Defensin S. cerevisiae Secreted Yeast pheromone mating [138]
factor α (MFα)
Lactoferrampin B Fragment of lactoferrin E. coli BL21 (DE3) Soluble Calmodulin [119]
LL-37 Cathelicidin E. coli BL21 (DE3) Soluble SmbP [41, 139]
Silk
SUMO
LsGRP1c Glycine Rich Protein from E. coli BL21 Soluble SUMO [140]
E. coli C41 (DE3)
E. coli C43 (DE3)
E. coli C41 (DE3) pLysS
E. coli C43 (DE3) pLysS
LvCrustinVII Crustacean AMP E. coli BL21(DE3) Solubilized – [141]
Magainin II F5W Cathelicidin E. coli BL21 (DE3) Soluble Calmodulin [119]
Magainin II-cecropin B Magainin/cathelicidin Cordyceps militaris Secreted Chimeric ­proteinb [50]
chimera hybrid
Melittin – E. coli BL21 (DE3) Soluble eGFP [117, 119]
Calmodulin
MIP-3α51-70 Chemokine fragment E. coli BL21 (DE3) Soluble Calmodulin [119]
MX226 Indolicidin derivative E. coli BL21(DE3) Soluble SUMO [41]
OrR214 and OrR935 Rice AMPs B. subtilis SCK Secreted – [142]
pBD-2-cecropin P1 chimera Defensin/cathelicidin B. subtilis Secreted Chimeric ­proteinb [143]
hybrid
Peptide P2 Designed peptide E. coli NM522 Solubilized Bovine prochymosin [144]
Pexiganan Magainin analogue E. coli BL21 (DE3) Soluble DAMP4 [145]
Pexiganan-honeybee Magainin analogue/silk- E. coli Rosetta 2 (DE3) Solubilized Silk [146]
silk chimera (modified fibre hybrid
magainin-2)
Plectasin Fungal defensin B. subtilis WB800N Secreted SUMO [147]
Plectasin Fungal defensin P. pastoris Secreted 4xPlectasin casette [46]
Porcine β-defensin 2 Porcine defensin E. coli BL21(DE3) Soluble – [141, 148]
(pBD-2)
PsDef5.1 Fungal defensin E. coli BL21(DE3) Codon‑ Soluble Thioredoxin (Trx) [149]
PlusRIL
Rosetta-gami 2(DE3)
Puroindoline A – E. coli BL21 (DE3) Soluble Calmodulin [119]
r(P)ApoBL, r(P)ApoBsa Encrypted ­peptidea E. coli BL21 (DE3) Solubilized Onconase [150]
rAvBD1-2–6–13 Chicken defensin L. lactis NZ3900 Soluble – [151]
Scorpine Defensin Anopheles gambie Secreted – [51]
Sericin-cecropin Silk-fibre/cathelicidin hybrid E. coli BL21 (DE3) Soluble Silk [152]
E. coli Rosetta (DE3)
Sesvania javanica defensin Defensin E. coli Origami 2 (DE3) Soluble Intein-CBD [153]
(Javanicin)
SMAP Cathelicidin E. coli BL21 (DE3) Soluble eGFP [117]
Snakin-1 (StSN1) Plant AMP Spodoptera frugiperda Secreted – [154]
(Baculovirus-infected insect
cells)
T9W Variant of pig myeloid anti‑ B. subtilis WB800N Secreted SUMO [155]
microbial peptide-36
Thanatin Insect AMP Human Embryonic Kidney Secreted – [156]
(HEK)293
Tilapia piscidin Piscidin P. pastoris Soluble – [44]
Tritrpticin Cathelicidin E. coli BL21 (DE3) Soluble Calmodulin [119]
Histidine tags were not considered to be fusion tags that help to express HDPs
Soluble: soluble protein produced in the cytoplasm; solubilized: protein solubilize from IBs; secreted: soluble protein secreted to the media
a
Denotes peptide fragment obtained from larger, non-antimicrobial proteins
b
We considered chimeric proteins those that do need cleaving of the tag as they add new and desired bioactivities

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Roca‑Pinilla et al. Microbial Cell Factories (2022) 21:267 Page 7 of 15

amounts of recombinant peptide loss due to degradation activity while showing a sustained release and a reduc-
[53]. Yields of HDPs produced in insect systems are less tion in pro-inflammatory cytokines release when com-
well documented than for yeast or bacterial systems, and pared to the non-encapsulated peptide [62]. Another
range between 6 and 25 mg/L [39]. approach that has been studied is the use of Polyethyl-
ene glycol (PEG)-stabilized lipodisks to protect cationic
Forms of recombinant HDPs peptides [63]. There is also a recent study that evaluated
In general, recombinant HDPs have been produced in 3 the encapsulated form of recombinant HDPs. Kaur and
main forms (Table 1): soluble protein (secreted or intra- coauthors analyzed the PEGylated form of mouse alpha-
cellular), inclusion bodies (IBs) or as encapsulated solu- defensin cryptdin-2 [64] and they observed a two-fold
ble protein. decrease in the antimicrobial activity when cryptidin-2
is conjugated to PEG. This effect could be attributed to a
Soluble recombinant production masking effect of PEG, and further studies are needed to
Many HDPs have been successfully obtained through evaluate the impact of PEGylation size and site, as previ-
recombinant production in different expression sys- ously described [64]. Going a step further, Drayton and
tems in a soluble form (Table 1) [54, 55]. In E. coli and coworkers have designed an enzyme-cleavable HDP-PEG
L. lactis, HDPs are typically produced intracellularly and system for the delivery of active HDPs, which is based
purified after cell disruption (Table 1). In contrast, when on the release of the antimicrobial peptide at the site
B. subtilis, yeast or fungi are used, the sequences of the of infection after cleavage by a host enzyme [65]. Thus,
proteins of interest are, mostly, designed to be secreted although much remains to be done, the results obtained
to the growth media (Table 1). However, in many other so far are promising when it comes to increase peptide
cases, especially when using bacterial expression systems, stability and decrease some of the adverse effects.
HDPs aggregate forming IBs, being necessary to solubi-
lize these protein aggregates (Table 1), as detailed in “IBs Antimicrobial inclusion bodies
as a source of soluble HDPs” section. IBs are protein nanoparticles or aggregates whose forma-
Besides aggregation, another issue of recombinant bac- tion has been widely described in E. coli [56, 57], but also
terial expression of HDPs is their potential lethality to the in other microbial expression systems such as lactic acid
recombinant host due to their antibacterial nature [56, bacteria [66–70] and yeast [70, 71]. These aggregates can
57]. In addition, they are highly susceptible to proteolysis be easily purified [72] and offer interesting features not
due to their small size and positive charge. To overcome available in a soluble form. IBs are an active biomaterial
all these problems, the most common strategy is to pro- that has already been explored in several applications
duce soluble HDPs with a fusion partner or to produce such as cancer [73], biocatalysis [74], tissue regenera-
them as multidomain proteins, as described in detail in tion [75] and immunostimulation [76], as they are highly
“Strategies to optimize HDP production” section. stable protein nanoparticles with slow-release properties
[56, 57, 77]. Recently, two studies have proven that HDP-
Encapsulated soluble HDPs based IBs are biologically active against different patho-
Some groups have also been working on the develop- genic bacteria [20, 37]. In the first study, López-Cano
ment of HDP delivery systems to have a time-controlled et al. showed that human α-defensin 5 (HD5) and lingual
release to improve bioavailability and to minimize tox- antimicrobial peptide (LAP) IBs are highly active against
icity and proteolytic degradation and, in consequence, MRSA and Pseudomonas aeruginosa, with antimicrobial
increase stability, when administered in vivo [58]. Most activities comparable to the soluble counterpart [37].
of these studies, however, have been done using synthetic The second study showed the antibiofilm properties of
HDPs. For example, different HDPs have been encap- IB-decorated surfaces against a carbapenem resistant
sulated using polymeric lactic-co-glycolic acid (PLGA) Klebsiella pneumoniae [20], adding to the evidence that
nanoparticles, showing that the encapsulated peptide antimicrobial IBs can be effectively used against AMR
kept the antimicrobial activity and did not increase bacteria.
toxicity when compared to the naked peptide [59, 60]. To maintain antimicrobial activity, constant admin-
Other studies using PLGA microspheres decorated with istration of an antimicrobial that has a short half-life is
N-acetyl cysteine (NAC) for pulmonary drug delivery required, as concentrations under minimum inhibitory
have shown to have a good potential both in vitro and concentrations (MIC) will probably happen during treat-
in vivo [61]. Synthetic HDPs have been also nanoencap- ment, further increasing the appearance of AMRs. This is
sulated in lipid-based nanoparticles. A peptide from the why a slow-release profile seems to be vital to maintain
cathelicidin family was encapsulated in liposomes and constant antimicrobial levels for long periods, to get an
the authors proved that the peptide kept its antimicrobial optimal therapeutic benefit, where HDP-based IBs are

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Roca‑Pinilla et al. Microbial Cell Factories (2022) 21:267 Page 8 of 15

promising protein format for antimicrobial applications denaturing protocols, this last strategy allows to obtain
[37]. Therefore, antimicrobial IBs can be used as nano- soluble protein from IBs without the need to use unfold-
pills that display antibacterial activity against different ing and refolding processes. Among these articles, some
AMR Gram-positive and Gram-negative strains. These of them have already successfully tried this approach
HDP-releasing nanopills can also be used to decorate using IBs formed by antimicrobial proteins [20, 21, 37].
plastic surfaces to avoid biofilm formation by bacteria Indeed, solubilized IB proteins show antimicrobial activ-
[20]. In addition, the IB format per se can be antimicro- ity against E. coli in a dose-dependent fashion against
bial, as one study found, where non-antimicrobial pro- carbapenem-resistant K. pneumoniae embedded in bio-
teins such as GFP and IFN-γ, when presented as IBs, films [20]. However, it is important to point out that the
achieved a significant reduction in bacterial loads [13]. selection of an optimal mild solubilizer is especially rel-
evant when antimicrobial proteins are purified because
IBs as a source of soluble HDPs recently it has been reported that they can impair the
IBs can also be used as an alternative source to obtain sol- antimicrobial activity [81].
uble HDPs when recombinant proteins aggregate (Fig. 3).
This is useful when the isolated soluble version of the Strategies to optimize HDP production
HDP is required for specific applications, but most of the Fusion tags
protein of interest forms aggregates. It is especially rel- There is a wide range of fusion partners (fusion tags) that
evant when HDPs are produced in E. coli, since in many have been used, as a strategy to properly express HDPs
cases it is necessary to recover the protein of interest in a soluble form (Table 1). These solubility enhanc-
from the aggregated fraction (Table 1). A high percentage ing domains assist with correct folding and promote the
(around 34%) of the recombinant HDPs produced in E. expression to the soluble fraction of the protein of inter-
coli are extracted from IBs (Table 1). est. In addition to enhance HDP solubility, tags might
In general terms, high concentrations of denatur- also protect against proteolysis, which HDPs are prone
ing agents such as 6M GdnHCl or 8M urea are used to to due to their small size [82]. Some widely used solu-
extract soluble protein from IBs. However, alternative bility tags that tend to yield high levels of HDPs in the
protocols have been developed in the last years [78]. cytoplasm of E. coli are GST, Trx, GFP, SUMO and Silk
Since it has been widely proven that proteins embedded (Table 1, Fig. 4A) [43, 83–88]. Although they need to be
in these nanoparticles can still be functional, the soluble studied empirically on a case to case basis, in general they
form of different proteins has also been extracted under considerably improve the solubility of many recombinant
mild, non-denaturing conditions [20, 66, 78–80]. Unlike proteins [89].

Fig. 3 Mild solubilization of HDP produced as IB in prokaryotic systems such as E. coli [20]

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Roca‑Pinilla et al. Microbial Cell Factories (2022) 21:267 Page 9 of 15

Fig. 4 Different strategies allow the successful production of HDPs, which might otherwise be refractory to recombinant production. A A
fusion partner that mimics the peptide precursor structure, but that must be removed downstream, B self-cleavable tags such as inteins, C
specificity-targeting tags and D a multi-domain HDP combined or not with antimicrobial proteins and other non-antimicrobial domains

An interesting approach is the use of the N-terminal Multidomain HDPs

domain of spider silk as a fusion tag, which has been Some researchers have explored the use of fusion part-
proven to yield up to 8 times more soluble protein ners that add other functions that go beyond just helping
compared to other frequently used expression tags and to fold and express the recombinant HDPs. An example
allows the expression of otherwise difficult to express of this are cationic elastin-like polypeptides (ELP), that
peptides and proteins, such as the HDP precursor of have successfully been used to purify a fusion hybrid of
LL-37 (hCAP18) [90]. Alternatively, acidic peptides can cecropin A and D without the need of chromatography
also be used, since they offer a charge-charge interac- [97]. ELP tags allow to form reversible spherical aggre-
tion that neutralizes the potential bactericidal effect of gates that allow to precipitate the fused HDPs under cer-
HDPs, avoiding the death of the host microorganism tain temperature conditions, in a process called inverse
[91]. transition cycling, thus simplifying downstream process-
However, in many cases, there is a need to remove the ing, in addition to allowing HDP expression.
carrier protein to isolate the peptide of interest. And A different example, that shows how fusion partners
often, removing the fusion partner requires expensive might add new features to HDPs, is the broad-spectrum
enzymatic cleavage or toxic reagents, such as cyanogen plant defensin HDP C6, which can be fused to a peptide
bromide or enterokinase hydrolysis [92, 93]. pheromone (cCF10) to add specificity to the original
To overcome tag removal hurdles, self-cleaving tags HDP. The cCF10 pheromone domain is species-specific
(Fig. 4B), such as inteins can be used. Inteins are protein and binds to the bacterial membrane of Enterococcus
segments that can cut themselves from their precursors faecalis with high affinity, an appealing addition to the
and re-join the flanking regions, also known as protein original antimicrobial activity that allows for the precise
splicing [94]. This system has been used to express a killing of E. faecalis while avoiding potential off-target
cathelicidin, using B. subtilis as a host, allowing to purify killing of beneficial microorganisms found in the host
the HDP by affinity chromatography and self-cleave in microbiome, which is what most the conventional anti-
one step [95]. Self-cleaving tags, therefore, enable puri- biotics do [98]. This type of multidomain approach is also
fication and cleavage in a single step, saving time, labor known as specifically targeted antimicrobial peptides
and reducing cost, but have an inherent risk of incom- (STAMPs) [99]. Many combinations of killing and tar-
plete or uncontrolled cleavage [96]. geting domains can be tried, where wild-type, rationally

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Roca‑Pinilla et al. Microbial Cell Factories (2022) 21:267 Page 10 of 15

enhanced or artificial sequences can be used [100–103]. individual components, namely catalytic, antimicrobial,
In all cases, these hybrid antimicrobials show improved and immune-modulatory activities [21, 104].
antimicrobial activity, selectivity, and kinetics against
their specific targets [87, 98]. Nonetheless, there still are Mutlidomain HDP fragment‑stitching
inevitable bactericidal effects on other bacteria. Like the multidomain approach, segments (but not the
The use of additional HDPs as fusion partners of full active sequence) of an HDP can be stitched together
other HDPs represents a unique strategy for the gen- in a hybrid molecule that is a mix of the parental pep-
eration of recombinant multidomain HDPs (Fig. 5D), tides, a process we named HDP fragment-stitching. Frag-
without the need for a carrier protein that needs to be ments of peptides such as the human cathelicidin LL-37,
removed downstream. For example, the pore-forming CM4 from a Chinese domestic silk moth and TP5, a frag-
HD5 has been fused to an enzyme that hydrolyses bac- ment of the thymus hormone, have been used in one
terial membrane phospholipids, such as human XII-A study to create hybrid antimicrobial that works against
secreted phospholipase A2 ­(sPLA2), generating a multi- enterotoxic E. coli [105]. Fragment-stitching might be
domain antimicrobial protein that can be successfully useful to remove undesired activities from a parental
expressed recombinantly, and that attacks bacteria by peptide, such as the hemolytic activity of LL-37, while
using two completely different mechanisms [20]. This generating new HDPs.
broad-spectrum multidomain construct, named JAMF1,
was effective against several antibiotic resistant bacterial Synergy of recombinant HDPs and antibiotics
strains such as quinolone and carbapenem resistant K. The performance of antibiotics against some antimicro-
pneumoniae (Fig. 5D). Similarly, in an effort to develop a bial resistant (AMR) bacteria or in bacterial living resist-
vaccine against glyceraldehyde-3-phosphate dehydroge- ant forms such as biofilms could be improved by their
nase (GAPDH), an enzyme involved in the virulence of combination with other drugs. Numerous studies have
Mycoplasma bovis, different bovine HDPs were fused to assessed this principle by using synthetic HDPs [106] and
GAPDH to boost the immune response against GAPDH antibiotics, demonstrating a clear synergy and beneficial
(i.e. BMAP28, TAP and indolicidin) [104]. Interest- effects on infection treatment. The main advantage of
ingly, when HDPs are linked to enzymes, such as in the this strategy is to reduce the dose of each drug and conse-
construct JAMF1 or GAPDH-HDP chimeras, both the quently the possible toxic effects and eventually combine
enzyme and the HDP seem to keep the activities of the different mechanisms to control bacterial survival along

Fig. 5 The boundless possibilities of multidomain HDPs. There is a vast range of domains to choose from for each domain we might want to add to
our chimeric HDP constructs, each one adding new functionalities, that might not necessarily be related to host defense (i.e. target specificity)

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Roca‑Pinilla et al. Microbial Cell Factories (2022) 21:267 Page 11 of 15

with the emergence of bacterial resistances. In the con- Abbreviations

ARB: Antimicrobial resistant bacteria; AMP: Antimicrobial peptide; HDP: Host-
text of recombinant HDPs, not many studies have been defense peptide; SPPS: Solid-phase peptide synthesis; PTMS: Post-translational
carried out. However, if synthetic HDPs can synergize modifications; GRAS: Generally Recognized as Safe; CHO: Chinese Hamster
with antibiotics, so should their recombinant version. Ovary; HSA: Human serum albumin; AMR: Antimicrobial resistance; rMBD:
Recombinant mouse beta defensin; MRSA: Methicillin resistant S. aureus; IB:
To explore this, researchers tested the synergy of mouse Inclusion bodies; HD5: Human alpha defensin 5; CAP: Cysteine rich acidic pep‑
β-defensin 3 (rMBD3) with different antibiotics against tide; MIC: Minimal inhibitory concentration; PLGA: Poly(lactic-co-glycolic acid);
bacterial and yeast drug-resistant strains in vitro [107]. NAC: N-Acetyl cysteine; PEG: Polyethylene glycol; hCAP: Human cathelicidin
antimicrobial protein; ELP: Elastin-like polypeptides; cCF10: Cell-associated
Interestingly, they found that the anti-methicillin-resist- pheromone peptide; STAMPS: Specifically targeted antimicrobial peptides;
ant S. aureus (MRSA) activity of rMBD3 in combination sPLA2: Secreted phospholipase ­A2; GAPDH: Glyceraldehyde 3-phosphate
with ampicillin was synergistic, but it was not effective dehydrogenase.
against methicillin sensible S. aureus [107]. Combina- Acknowledgements
tions of rMBD3 with itraconazole, amphotericin or 5-flu- Figures were created with BioRender.com.
orocytosine were synergistic against two tested Candida
Author contributions
albicans strains. These results support the potential of RRP, AA and EGF conceived and designed the manuscript. RRP, AA and EGF
recombinant HDP to improve the activities of conven- performed the bibliographic research, conceptualized, and drafted the manu‑
tional antibiotics [107] and suggest that the same mecha- script. LL outlined the structure and reviewed the manuscript. All authors read
and approved the final manuscript.
nism that makes bacteria resistant to a certain antibiotic
might make them more vulnerable to combinatorial Funding
treatments. This work was funded by Ministerio de Ciencia, Innovación y Universidades
Grant (PID2019-107298RB-C21/AEI/10.13039/501100011033) to AA and EG-F
and by Marató de TV3 foundation (201812-30-31-32-33) to EG-F. The authors
Conclusions are also indebted to CERCA Programme (Generalitat de Catalunya) and Euro‑
pean Social Fund for supporting our research. RRP was supported by internal
HDPs hold promise as new candidates to combat anti- funding from Children’s Medical Research Institute.
biotic-resistant pathogens. There is a clear need for
new potent HDP candidates and the means to produce Availability of data and materials
The datasets supporting the review are referenced throughout the article.
them efficiently and at a low-cost. Research on different
expression systems will effectively accelerate our ability
Declarations
to increase production yields, peptide structure and bio-
activity and provide access to engineered microbial cell Ethics approval and consent to participate
factories that work universally well for most HDPs. But Not applicable.

the optimal expression systems are only one part of the Consent for publication
equation. Not applicable.
New recombinant HDP forms, such as IBs and encap-
Competing interests
sulated HDPs, are also worth considering. They offer The authors have no competing interests to disclose.
properties that the soluble form does not provide, includ-
ing higher stability, a slower release profile, reduced HDP Author details
1
Translational Vectorology Research Unit, Faculty of Medicine and Health, Chil‑
toxicity or on-site activation. Besides, IBs should be dren’s Medical Research Institute, The University of Sydney, Westmead, NSW
exploited as both a new antimicrobial HDP format and 2145, Australia. 2 Laboratory of Molecular Oncology and Innovative Therapies,
as a treasure trove of bioactive, functional, and soluble Military Institute of Medicine, Warsaw, Poland. 3 Department of Ruminant Pro‑
duction, Institut de Recerca i Tecnologia Agroalimentàries IRTA​, 08140 Caldes
HDPs. de Montbui, Spain.
Finally, strategies to optimize HDP production, such
as fusion tags and multidomain HDPs, open the door to Received: 7 November 2022 Accepted: 10 December 2022

designing newly added functionalities, such as the pre-

cise killing of a pathogen, while avoiding off-target effects
on probiotic bacteria. Moreover, a well-crafted tag strat-
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